Functional Requirement for Orai1 in Store-operated TRPC1-STIM1 Channels*

Orai1 and TRPC1 have been proposed as core components of store-operated calcium release-activated calcium (CRAC) and store-operated calcium (SOC) channels, respectively. STIM1, a Ca2+ sensor protein in the endoplasmic reticulum, interacts with and mediates store-dependent regulation of both channels. We have previously reported that dynamic association of Orai1, TRPC1, and STIM1 is involved in activation of store-operated Ca2+ entry (SOCE) in salivary gland cells. In this study, we have assessed the molecular basis of TRPC1-SOC channels in HEK293 cells. We report that TRPC1+STIM1-dependent SOCE requires functional Orai1. Thapsigargin stimulation of cells expressing Orai1+STIM1 increased Ca2+ entry and activated typical ICRAC current. STIM1 alone did not affect SOCE, whereas expression of Orai1 induced a decrease. Expression of TRPC1 induced a small increase in SOCE, which was greatly enhanced by co-expression of STIM1. Thapsigargin stimulation of cells expressing TRPC1+STIM1 activated a non-selective cation current, ISOC, that was blocked by 1 μm Gd3+ and 2-APB. Knockdown of Orai1 decreased endogenous SOCE as well as SOCE with TRPC1 alone. siOrai1 also significantly reduced SOCE and ISOC in cells expressing TRPC1+STIM1. Expression of R91WOrai1 or E106QOrai1 induced similar attenuation of TRPC1+STIM1-dependent SOCE and ISOC, whereas expression of Orai1 with TRPC1+STIM1 resulted in SOCE that was larger than that with Orai1+STIM1 or TRPC1+STIM1 but not additive. Additionally, Orai1, E106QOrai1, and R91WOrai1 co-immunoprecipitated with similar levels of TRPC1 and STIM1 from HEK293 cells, and endogenous TRPC1, STIM1, and Orai1 were co-immunoprecipitated from salivary glands. Together, these data demonstrate a functional requirement for Orai1 in TRPC1+STIM1-dependent SOCE.

Store-operated Ca 2ϩ entry (SOCE) 3 is mediated via activation of specific plasma membrane channels in response to depletion of Ca 2ϩ from intracellular Ca 2ϩ stores (1). Neither the mechanism by which the status of Ca 2ϩ in the endoplasmic reticulum is transmitted to the plasma membrane nor the molecular components of the channels have yet been conclusively identified in all cell types. Several reports suggest a diversity in store-operated Ca 2ϩ channels in different cell types (2)(3)(4). For example, calcium release-activated calcium (CRAC) channel, which is found in T-lymphocytes, RBL, and other hematopoietic cells, is a highly Ca 2ϩ -selective channel with unique properties (4,5). Channels in other cell types, including salivary gland, endothelial, and smooth muscle cells, referred to as SOC channels, range from non-selective to relatively Ca 2ϩselective (2-4, 6, 7). It is believed that the difference in channel property is due to differences in the channel components.
TRPC1 is reported to form SOC channels that range from being relatively selective for Ca 2ϩ to those that are non-selective in various cell types (2,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). With the exception of a few studies (17,18), TRPCs do not appear to generate I CRAC . Consistent with this, we have shown that TRPC1 does not contribute to the I CRAC in RBL-2H3 cells (19). Two proteins, STIM and Orai, have emerged as candidate components of the CRAC channel (5,20). Knockdown of STIM1 expression using siRNA significantly reduced SOCE in a number of cell types (20 -23), whereas overexpression only modestly enhanced SOCE. The second protein Orai1 has four transmembrane domains (5,20,24). Mutations in Orai1 have been genetically linked to severe combined immunodeficiency (SCID), and T-lymphocytes isolated from SCID patients display decreased I CRAC activity (20). Although knockdown of Orai1 decreases SOCE, overexpression of the protein attenuates endogenous SOCE. However, coexpression of Orai1 with STIM1 increases SOCE and generates CRAC channel activity in HEK293 cells (25,26). Further, mutations in the conserved negatively charged residues of Orai1 alter the Ca 2ϩ selectivity of CRAC channel (27,28). Thus, it has been suggested that Orai1 and STIM1 are sufficient for the formation of CRAC channel and that Orai1 is the pore-forming unit. The contribution of Orai proteins to SOCE in all cell types is not yet clear. Not all cells that express Orai proteins demonstrate CRAC currents (24,29,30). A recent report demonstrated that Orai1 does not form the CRAC channel in mouse T-lymphocytes (31). Thus, it has been suggested that Orai and STIM proteins might serve multiple functions and display very different biophysical properties in different cell types depending on the molecular composition of the channel complexes. Orai channel complexes might consist of not only different Orais and STIMs but also other channel subunits (24). The possibility of Orai1 being associated with a larger protein complex was also suggested (32).
We have previously reported that dynamic assembly of a TRPC1-STIM1-Orai1 complex is involved in SOCE in salivary gland cells (19). We have shown that endogenous Orai1, STIM1, and TRPC1 concertedly regulate SOCE in these cells. Although heterologous expression of TRPC1 alone does not increase SOCE, co-expression with STIM1 was reported to increase SOCE and SOC channel function in HEK293 cells (33,34). Further, a suggestion has been made that Orai1 might function as a regulator of TRPC3 and TRPC6, conferring store-dependent activation of these channels (35). In this study, we have examined the molecular basis of TRPC1-dependent SOC channels. We report that in addition to STIM1, there is a functional requirement for Orai1 in the generation of TRPC1-SOC channels.

EXPERIMENTAL PROCEDURES
HEK293 Cell Culture and Transfection-HEK293 cells were grown in Dulbecco's modified Eagle's medium and 10% heatinactivated fetal bovine serum supplemented with 100 units/ml penicillin G and 100 g/ml streptomycin. Cells were allowed to grow to ϳ70% confluence and transfected with required DNA at concentration of 1 g/ml, using Lipofectamine 2000 and protocols supplied by the manufacturer (Invitrogen). Knockdown experiments were carried out by transfection of Orai1 siRNA (sequence number ucacugguuagccauaaga) or control siRNA (Dharmacon, Chicago, IL), transfected at a concentration of 0.8 nmol/ml, using DharmaFECT Duo reagent and protocols supplied by the manufacturer.
Electrophysiology-Coverslips with HEK cells were transferred to the recording chamber and perfused with an standard external solution with the following composition (in mM): NaCl, 145; KCl, 5; MgCl 2 , 1; CaCl 2 , 1; Hepes, 10; glucose, 10; pH 7.4 (NaOH). The patch pipette had resistances between 3 and 5 milliohms after filling with the standard intracellular solution that contained the following (in mM): cesium methane sulfonate, 145; NaCl, 8; MgCl 2 , 10; Hepes, 10; EGTA, 10; pH 7.2 (CsOH). Osmolarity for all the solutions was adjusted with mannose to 300 Ϯ 5 mosM using a vapor pressure Osmometer (Wescor, Logan, UT). Whole cell patch clamp experiments were performed in the standard whole cell configuration at room temperature (22-25°C) using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). Generation of the current was assessed by the amplitude at Ϫ80 mV, taken from the currents recorded during voltage ramps ranging from Ϫ90 to 90 mV over a period of 1 s imposed every 4 s (holding potential was 0 mV) and digitized at a rate of 1 kHz. Liquid-junction potentials were less than 8 mV and were not corrected. Capacitative currents and series resistance were determined and minimized. For analysis, current recorded during the first ramp was used for leak subtraction of the subsequent current records.
[Ca 2ϩ ] i Measurements-Fura2 fluorescence was measured in single HEK cells cultured for 24 -48 h in glass bottom MatTek tissue culture dishes (MatTek Corp. Ashland, MA) and transfected as required. Fluorescence was recorded in Fura2-loaded cells using a Till Photonics-Polychrome V spectrofluorimeter and MetaFluor imaging software (Molecular Devices). Student's t test was used to statistically evaluate the data.

RESULTS AND DISCUSSION
Expression of Orai1ϩSTIM1 Increases SOCE and Generates I CRAC - Fig. 1, panel i, shows that expression of Orai1 and STIM1 in HEK293 cells induces a significant increase in thapsigargin (Tg)-stimulated SOCE (Fig. 1, panel ii). Expression of STIM1 alone did not induce any change in SOCE, whereas expression of Orai1 induced a slight inhibition of SOCE (data not shown). Expression of Orai1ϩSTIM1 resulted in activation of typical inwardly rectifying I CRAC -like currents in response to Tg stimulation of cells ( Fig. 1, panels iii and iv) as has been previously reported (25,26).
TRPC1-dependent SOCE Is Dependent on STIM1 and Orai1 in HEK293 Cells-Although co-expression of Orai1 and STIM1 in HEK293 cells is reported to generate the CRAC channel (5), expression of TRPC channels with STIM1 has been shown to generate SOC channels in these cells (33)(34)(35). How exactly these two distinct channels are assembled and whether any endogenous proteins are involved in the process is not yet known. Fig. 1, panel v (see panel viii for average data), shows that siOrai1 induced 80% decrease in endogenous SOCE (transfection with control siRNA did not affect SOCE when compared with non-transfected cells, trace not shown). siSTIM1 also significantly reduced SOCE (data not shown). The decrease in endogenous Orai1 by siOrai1 is shown in the inset (lane 1 shows Orai1 in cells treated for 48 h with control siRNA, and lanes 2, 3, and 4 show the protein after 48 h of treatment with 0.2, 0.4, and 0.8 nmol of siOrai1, respectively. Actin expression in the samples is shown as a control. Note that 0.8 nmol of siOrai1 was used in the experiments described here). Overexpression of TRPC1 induced a 1.75-fold increase in SOCE (Fig. 1, panel vi), whereas overexpression of TRPC1ϩSTIM1 induced 2.4-fold increase in SOCE relative to that in control cells (Fig. 1, panel vii). Ca 2ϩ entry in these cells was blocked by 1 M Gd 3ϩ as well as by 20 M 2-APB (supplemental Fig. 1), showing that TRPC1ϩSTIM1 primarily result in SOCE. Importantly, knockdown of endogenous Orai1 attenuated TRPC1-or TRPC1ϩSTIM1-induced SOCE by Ͼ50% (i.e. the increase in 340/380 ratio in TRPC1ϩSTIM1 cells is about 0.7, whereas that in siOrai1ϩTRPC1ϩSTIM1-expressing cells is 0.35. Note that these values represent the increase in 340/380 ratio above that in control cells following Ca 2ϩ readdition to cells, i.e. due to Ca 2ϩ entry). Thus, knockdown of endogenous Orai1 decreased STIM1-dependent increase in SOCE in TRPC1-expressing cells (SOCE in TRPC1ϩSTIM1 cells was 1.2 when compared with 0.7 in cells expressing TRPC1 alone, i.e. the increase due to STIM1 is 0.5. With expression of siOrai1 in both sets of cells, this increase was attenuated to 0.2, a 60% decrease, Fig. 1, panel vii). Thus, endogenous Orai1 is required for exogenously expressed TRPC1 and STIM1 to increase SOCE. The data also show that the increase in TRPC1 function conferred by STIM1 is dependent on endogenous Orai1.
The contributions of TRPC1, STIM1, and Orai1 in SOCE were further examined by measuring store-operated currents.
In contrast to the current seen with Orai1ϩSTIM1 ( Fig. 1, panels iii and iv), cells expressing TRPC1ϩSTIM1 displayed linear currents following Tg stimulation, which could be blocked by 20 M 2-APB ( Fig. 1, panel ix and x, similar inhibition was seen with 1 M Gd 3ϩ , data not shown). Note that about 40% of the cells displayed spontaneous currents, which in some cells could be further increased with Tg. In both cases, the current was fully blocked by 1 M Gd 3ϩ (supplemental Fig. 1). Together, these data demonstrate that TRPC1ϩSTIM1 generate SOC channels that are distinct from CRAC channels in the property of their currents. The characteristics of TRPC1ϩSTIM1 channels in these cells are similar to those described by Yuan et al. (34). Importantly, TRPC1ϩSTIM1-induced I SOC was significantly reduced by knockdown of endogenous Orai1 (Fig. 1, panels xi and xii, and see Fig. 2, panel x, for average data). Thus, TRPC1ϩSTIM1-dependent SOC channel function also requires endogenous Orai1.
Together, the data shown in Fig. 1 demonstrate that TRPC1 requires overexpression of STIM1 to generate SOC channels in HEK293 cells. This most likely explains why in previous studies there was no increase in function associated with TRPC1 overexpression in these cells (4). A novel finding of the present study is that the ability of TRPC1 and STIM1 to increase SOCE and generate SOC channels is dependent on endogenous Orai1. We detected a small contribution of TRPC1 to endogenous SOCE in HEK293 cells, and shTRPC1 induced an ϳ20% decrease. However, shTRPC1 did not affect Orai1ϩSTIM1-dependent increase in SOCE and I CRAC (data not shown). Consistent with these findings, we have previously shown that endogenous TRPC1 does not contribute to I CRAC in RBL-293 cells (19). Several recent studies have also suggested lack of a role for TRPC1 in Orai1ϩSTIM1-generated CRAC channels in HEK293 cells (25,26). Thus, the molecular requirement for generation of Orai1ϩSTIM1-CRAC channels appears to be different from those of TRPC1ϩSTIM1-SOC channels in this cell type. We have previously demonstrated an association between TRPC1-STIM1-Orai1 in human submandibular gland (HSG) cells. The present data would suggest that exogenously expressed STIM1 and TRPC1 associate with endogenous Orai1 to form a functional SOC channel in HEK293 cells. Unfortunately, the latter observation could not be determined biochemically due to the lack of an appropriate Orai1 antibody.
Functional Orai1 Is Required for TRPC1ϩSTIM1-dependent SOCE-Severe loss of I CRAC in T-lymphocytes isolated from SCID patients has been linked to a mutation (R91W) near the first TM domain of Orai1. The mutant channel has been shown to be inactive (20). Although exactly why this mutant is not functional is not yet understood, it is interesting that a conserved sequence in the N terminus of Orai1, amino acids 74 -90 immediately upstream of the mutation site, has been suggested to be involved in activation of the channel (36). Expression of R91WOrai1 in HEK293 cells induced a decrease in endoge- nous SOCE (Fig. 2, panel ii, trace not shown). Expression of TRPC1ϩSTIM1 in cells expressing R91WOrai1 resulted in SOCE similar to that seen in cells expressing TRPC1ϩSTIM1ϩsiOrai1 (Fig. 2, panel i and ii, compare with Fig. 1, vii and viii). Similar inhibition of endogenous SOCE (trace not shown, Fig. 2, panel ii) as well as SOCE in TRPC1ϩSTIM1-expressing cells was seen by expression of E106QOrai1 (Fig. 2, panels i and ii). This mutant of Orai1 has decreased ion permeability and has been shown to exert dominant suppression of endogenous SOCE as well as Orai1ϩSTIM1-dependent SOCE in HEK293 cells (27,28). In contrast, expression of Orai1 together with TRPC1ϩSTIM1 induced SOCE that was larger than that seen with TRPC1ϩSTIM1 or Orai1ϩSTIM1, but not additive (increase was calculated relative to SOCE in control cells, Fig. 2i, average data shown in panel ii). Expression of TRPC1ϩOrai1 without STIM1 did not change SOCE (data not shown). Fig. 2, panel iii, shows that R91WOrai1, E106QOrai1, and Orai1 (all FLAG-tagged) were expressed at similar levels and co-immunoprecipitated with comparable levels of STIM1 as well as TRPC1 (note that none of the proteins were immunoprecipitated in control IPs using anti-FLAG antibody and lysates of non-transfected HEK cells, supplemental Fig. 2). Thus, the functional differences seen in cells expressing the wild type and mutant proteins are not due to differences in their expression levels or their ability to associate with STIM1 or TRPC1. Further, IP of TRPC1 pulls down similar level of STIM1 in cells where Orai1 was not overexpressed (data not shown), indicating that there is no disruption of STIM1-TRPC1 association by expression of Orai1 (also see Ref. 19). The association between the three proteins was also observed in submandibular gland cells. IP of endogenous TRPC1 co-immunoprecipitated endogenous Orai1 and STIM1 (Fig. 2, panel iii,  right blot). These data provide strong evidence that there is close association between TRPC1, Orai1, and STIM1.
The requirement of functional Orai1 was assessed by using functionally deficient Orai1 mutants. Co-expression of R91WOrai1 (the mutant in SCID patients) or E106QOrai1 (the pore-deficient mutant) with TRPC1ϩSTIM1 induced Ͼ70% inhibition of I SOC (Fig. 2, panels vi to ix, respectively, also see Fig. 2, panel x, for average data). Since siOrai1 also induces similar attenuation of TRPC1ϩSTIM1-dependent SOC channel activity, it is unlikely that competition for STIM1 accounts for the observed attenuating effect of the mutant Orai1 proteins on TRPC1ϩSTIM1-SOC function. In contrast, co-expression of Orai1 with TRPC1ϩSTIM1 (Fig. 2, panel iv and v) induced non-selective linear current in 8/11 cells, which was 30% larger in amplitude than that seen with TRPC1ϩSTIM1 or Orai1ϩSTIM1 (Fig. 2, panels iv, v, and x). In 3/11 cells, the currents were weakly inwardly rectifying but relatively nonselective, i.e. more like I SOC . Note that when Orai1-cDNA was used during transfection was increased (5 g instead of 1 g), 3/7 cells displayed linear non-selective currents, 2/7 displayed I SOC -like currents with right shift in the E rev , and 2/7 displayed I CRAC -like currents (data not shown).
The data reported above demonstrate that co-expression of STIM1 with TRPC1 is required for generation of SOC channels in HEK293 cells. This is similar to the STIM1 requirement reported for the generation of CRAC channels in HEK293 cells by exogenous expression of Orai1 (5). Further, we show that TRPC1ϩSTIM1 generate channels that display characteristics that are distinct from Orai1ϩSTIM1-generated CRAC channels. These data are consistent with previous studies describing TRPCϩSTIM1-dependent SOC channel activity in HEK293 cells (33,34). Together, these data also account for the previously reported lack of effect of heterologously expressed TRPC1 on SOCE.
An important and novel finding of the present study is that endogenous Orai1 is required for TRPC1ϩSTIM1-generated SOC channel function. Further, non-functional Orai1 mutant, R91WOrai1, or the permeability-defective mutant, E106QOrai1, attenuate the function of TRPC1ϩSTIM1-SOC channels, whereas Orai1 increases TRPC1ϩSTIM1-dependent SOCE. Although we do not yet understand how exactly Orai1 contributes to TRPC1ϩSTIM1 channel function, possible mechanisms that can be proposed are: (i) TRPC1 and Orai1 contribute to the same channel, (ii) Orai1 and TRPC1 form distinct channels, whereby the function of Orai1 somehow regulates TRPC1, and (iii) TRPC1 and Orai1 form distinct and independent channels. Although the present data do not exclude the first two possibilities, several of our observations, described here and previously (19), suggest that when co-expressed, TRPC1 and Orai1 do not form distinct and independent channels. First, increase in the current as well as Ca 2ϩ entry obtained in cells expressing Orai1ϩSTIM1ϩTRPC1 (increase in 340/380 ratio above control ϭ 0.9) is less than that expected if Orai1ϩSTIM1 and TRPC1ϩSTIM1 generated separate channels (together the increase in 340/380 ratio should be 1.4). We do not believe that this is due to a competition between TRPC1 and Orai1 for STIM1 since transfection with different amounts of STIM1 cDNA gave similar results (data not shown). Also, co-IP of exogenously expressed TRPC1 and STIM1 was not affected by co-expression of Orai1 (19). Finally, siOrai1 did not increase TRPC1ϩSTIM1-mediated SOCE as would be expected if Orai1 were competing for a limited pool of STIM1. Although more studies are required to determine the precise functional association between TRPC1 and Orai1, based on our data, we propose that the proteins converge on the same SOC channel. Orai1 has been shown to contribute to the ion permeability of CRAC channel, which has been reported to be independent of TRPC1 and other TRPC proteins. However, a recent study showed that several neuronal cells express Orai proteins but do not display I CRAC (24), and it was suggested that Orai proteins might interact with each other or with other proteins to form diverse SOC channels. An interesting role for Orai1 proposed by Liao et al. (35) was that it serves as a regulatory subunit for TRPC3 and TRPC6 channels and confers coupling to the store by mediating the regulation by STIM1. Our previous study suggested a possible association between Orai1 and TRPC1 in cells expressing SOC channels (19). Here we have shown that functional Orai1 is required for generation of TRPC1-SOC channel. These data do not exclude the possibility that Orai1 and TRPC1 form the same SOC channel, although how this is achieved will need to be addressed in future studies. Alternatively, Orai1-CRAC channels might somehow regulate TRPC1-SOC channels. Although the exact contribution of Orai1 in TRPC1-dependent SOC channel function has yet to be determined, we have shown above that the three proteins are closely associated endogenously in salivary glands where TRPC1 has a major role in SOCE (19).
In conclusion, store-operated Ca 2ϩ entry appears to be mediated via distinct channels in different cell types. Although some channels are OraiϩSTIM-based, others depend on TRPCϩSTIM. We recently reported that TRPC1 Ϫ/Ϫ salivary gland cells had greatly reduced SOCE and I SOC , which was associated with a significant and stable loss of fluid secretion, although all three Orai transcripts were detected in TRPC1 Ϫ/Ϫ cells (37). Thus, the Orai proteins were unable to compensate for the loss of TRPC1. Here we show that TRPC1 generates SOC channels in a STIM1-dependent manner and that these channels are distinct from CRAC channels. Importantly, our data demonstrate a novel functional requirement for Orai1 in TRPC1-generated SOC channel. Thus, STIM1 and Orai1 appear to be required for the generation of CRAC as well as SOC channels. Although Orai1ϩSTIM1 appear to be sufficient for CRAC channels, TRPC1, Orai1, and STIM1 concertedly generate SOC channels. Further studies are required to determine the exact molecular interactions between these proteins that determine SOCE.